Erythropoietin compositions

ABSTRACT

Methods and materials are provided for the production of compositions of erythropoietin protein, wherein said compositions comprise a pre-selected N-linked glycosylation pattern as the predominant N-glycoform.

RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.11/804,510, filed May 18, 2007, now U.S. Pat. No. 7,851,438, whichclaims priority to provisional applications U.S. Ser. No. 60/801,688,filed May 19, 2006 and U.S. Ser. No. 60/905,770, filed Mar. 7, 2007,each of which are hereby incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name GFIBIO22376USDIV_SEQTXT_(—)13MAY2010.TXT”, creation date ofMay 13, 2010, and a size of 3 KB. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, inparticular the invention provides materials and methods for theproduction of homogeneous compositions of PEGylated erythropoietin withdesired N-glycoforms.

BACKGROUND OF THE INVENTION

The production of recombinant glycoproteins has been an area of greatactivity for the biotechnology industry. One drawback of recombinantglycoproteins has been the heterogeneity of glycosylation produced bycommonly used cellular host such as CHO cells. In contrast, the presentinvention provides lower eukaryotic host cells that have been engineeredto produce recombinant erythropoietins comprising pre-selected desiredN-glycan structures. The compositions of recombinant erythropoietinsproduced therefrom are significantly greater in uniformity of glycoformsthan those produced in CHO cells.

Erythropoietin is a protein hormone which has been widely used fortherapeutic indications requiring increased formation of red blood cellsincluding anemia due to renal failure or chemotherapy treatment. Becauseof the great demand for safe and effective erythropoietin, recombinanthuman erythropoietin has become the largest selling recombinant humanprotein product.

Native human erythropoietin contains four carbohydrate chains (threeN-linked and one O-linked). The protein requires tri- andtetra-antennary sialylated N-glycans for maximum in vivo efficacy.However, in vitro receptor binding and cell-based assays reveal thaterythropoietins with multi-branched sialylated glycans anderythropoietins with additional N-glycosylation sites exhibit decreasedbinding relative to enzymatically deglycosylated erythropoietin. Thisparadox can be explained by considering clearance from the circulatorysystem. Tetra-antennary sialylated erythropoietin and darbepoetinexhibit longer serum half-lives compared with bi-antennary sialylatedand nonglycosylated erythropoietin. (The principal routes of clearancefor erythropoietin are via renal filtration, through binding to theasialoglycoprotein receptor, endothelial cell uptake and internalizationby the target cell through the erythropoietin receptor.)

Currently marketed forms of recombinant erythropoietin include Epogenwith three tetra-antennary N-glycan structures and Aranesp,erythropoietin engineered to contain two additional N-glycosylationsites for a total of five tetra-antennary N-glycan structures. Theaddition of these extra N-glycan attachment sites has resulted in alonger serum half-life and consequently an increased in vivo activity ofthe hormone. These erythropoietins are produced from CHO cells andsecreted with a heterogeneous mixture of N-glycan structures. Processdevelopment is used to enrich for the tetra-antennary sialylatedglycoform (see FIG. 1).

Past efforts to improve upon the properties of erythropoietin haveincluded efforts to alter the glycosylation, for example by addingglycosylation sites, as well as efforts to conjugate the protein topolymers, such as polyethylene glycols. See, for example EP 0640619; WO00/32772 WO 01/02017 and WO 03/029291. Despite these attempts, thereremains a need for recombinant erythropoietin having improved propertiessuch as greater ease of administration; less rigorous dosage regimens;improved pharmacokinetics and bioavailability; and less expensivemanufacture. In particular, robust processes and materials useful toproduce compositions of erythropoietin possessing these qualities fromlower eukaryotic cells has remained an elusive and desirable goal.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and materials forproduction of vectors and host cells which are capable of expressingrecombinant human erythropoietin with specifically directedN-glycosylation, as well as compositions of recombinantly engineeredglycoproteins which have been chemically modified, for example, byPEGylation.

The present invention provides engineered strains of lower eukaryotes,particularly, Pichia pastoris, which are capable of producing fullysialylated recombinant EPO with bi-antennary N-glycan structures.

The present invention also provides a PEGylated EPO with bi-antennaryglycans. These recombinant glycoprotein compositions exhibit theenhanced in vivo bio-activity previously seen in vitro while maintainingincreased serum half-life

Thus, the present invention provides methods and materials for theproduction of compositions of PEGylated erythropoietin protein, saidcomposition comprising a plurality of N-linked glycoforms comprising atleast one N-linked glycan attached thereto. In preferred embodiments thecomposition comprises a plurality of N-linked glycans in which greaterthan 25 mole percent of said plurality of N-linked glycans consistsessentially of a desired N-linked glycan structure selected from thegroup consisting of:

GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂; GlcNAc₂Man₃GlcNAc₂Gal₂; GlcNAc₂Man₃GlcNAc₂;

GlcNAc₂Man₃; GlcNAc₂Man₅GlcNAcGalNANA; GlcNAc₂Man₅GlcNAcGal;

GlcNAc₂Man₅GlcNAc; and GlcNAc₂Man₅.

In further preferred embodiments, the desired N-linked glycans structurecomprises greater than 50 mole percent; 75 mole percent or 80 molepercent of said N-linked glycan structures.

In one preferred embodiment, the desired N-linked glycan structureconsists essentially of GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.

In further preferred embodiments, the PEGylated erythropoietin proteincomposition comprising a plurality of glycoforms, each glycoformcomprising at least one N-linked glycan attached thereto, wherein theglycoprotein composition thereby comprises a plurality of N-linkedglycans in which greater than 25 mole percent of said plurality ofN-linked linked glycans consists essentially ofGlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.

In further preferred embodiments, the present invention comprisescompositions in which greater than 50 mole percent; 75 mole percent or80 mole percent of said plurality of N-linked glycans consistsessentially of GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.

In further preferred embodiments, the present invention comprisescompositions of PEGylated erythropoietin in which theGlcNAc₂Man₃GlcNAc₂Gal₂NANA₂ N-linked glycan is present at a level fromabout 5 mole percent to about 80 mole percent more than the next mostpredominant N-linked glycan structure of said plurality of N-linkedglycans.

Another preferred glycoform of the present invention consistsessentially of the structure GlcNAc₂Man₃GlcNAc₂Gal₂. In preferredembodiments, the PEGylated erythropoietin protein composition comprisinga plurality of glycoforms, each glycoform comprising at least oneN-linked glycan attached thereto, wherein the glycoprotein compositionthereby comprises a plurality of N-linked glycans in which greater than25 mole percent of said plurality of N-linked linked glycans consistsessentially of GlcNAc₂Man₃GlcNAc₂Gal₂.

In further preferred embodiments, the present invention comprisescompositions in which greater than 50 mole percent greater than 75 molepercent of said plurality of N-linked glycans consists essentially ofGlcNAc₂Man₃GlcNAc₂Gal₂.

In further preferred embodiments, the present invention comprisescompositions of PEGylated erythropoietin in which theGlcNAc₂Man₃GlcNAc₂Gal₂ N-linked glycan is present at a level from about5 mole percent to about 80 mole percent more than the next mostpredominant N-linked glycan structure of said plurality of N-linkedglycans.

In preferred compositions of the present invention, a polyethyleneglycol moiety and the N-terminal amino acid residue of erythropoietinprotein may be linked, either directly or via a linker.

In preferred embodiments of the invention, the polyethylene glycolmoiety may have a molecular weight of from about 5 to about 100 kD; morepreferably about 20 kD to about 60 kD; weight of from about 30 kD toabout 40 kD.

The present invention also provides therapeutic methods employing therecombinant erythropoietins. In preferred embodiments the recombinanterythropoietin is administered to a human patient in need of treatmentto alleviate an anemia. In further preferred embodiments, the patient istreated for anemia induced by renal failure, chemotherapy or cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D illustrates the stepwise creation of the P. pastoris strainYGLY3159 that secretes human recombinant EPO on which the predominantN-glycan is a fully sialylated glycan, represented as:GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.

FIG. 2 Panels A-O show maps of the plasmid vectors referred to in FIG. 1that were used to generate YGLY3159.

FIG. 3 illustrates a three-step chromatographic separation process usedto purify human EPO.

FIGS. 4A-4D shows the characterization of purified EPO by Panel 4A)SDS-PAGE, showing overall purity of sample Panel 4B) size exclusionchromatography (SEC-HPLC) showing a uniform single peak demonstratingthe lack of degradation and lack of aggregation Panel 4C) reverse phase(RP)-HPLC demonstrating intactness of EPO and Panel 4D) LC-MSdemonstrating product quality on PNGase F treated EPO.

FIG. 5 shows SDS PAGE analysis of the purified hEPO sample conjugated toa 30 kDa, 40 kDa, or 60 kDa linear PEG or a 45 kDa branched PEG.

FIG. 6 shows size exclusion chromatography (SEC HPLC) analysis of fourdifferent PEGylated EPO products.

FIG. 7 shows high performance liquid chromatograms of the N-linkedglycans released from unPEGylated EPO and four different PEGylated EPOproducts by treatment with PNGase-F and labeled with 2-aminobenzidine(2-AB) The chromatograms demonstrate the predominance of thebisialylated glycoform GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.

FIG. 8 shows that mice injected with four different versions of PEG-EPOconjugates display an increase in hematocrit over commercially purchasedAranesp. C57B6 mice were dosed twice weekly for a total of fiveinjections (dosing was stopped after 2.5 weeks). The mice were bledweekly and hematocrit values were determined.

FIGS. 9A-9B show preferred N-linked glycoforms of erythropoietin whichmay be produced in accordance with the present invention.

FIG. 10 shows a overview of a purification strategy for recombinanthuman EPO.

FIG. 11 shows a Coomassie stained gel of purified recombinant human EPO.

FIG. 12 shows schematic representations of PEGylation chemistries.

FIG. 13 shows the effect of once weekly PEG-EPO dosing on relativehematocrit increase in mice.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQUENCE ID NO: 1 A DNA sequence encoding a human erythropoietin.

SEQUENCE ID NO: 2 Amino acid sequence encoding a human erythropoietin.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the present invention shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular.Generally, nomenclatures used in connection with, and techniques ofbiochemistry, enzymology, molecular and cellular biology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer,Introduction to Glycobiology, Oxford Univ. Press (2003); WorthingtonEnzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbookof Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbookof Biochemistry: Section A Proteins, Vol II, CRC Press (1976);Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

All publications, patents and other references mentioned herein arehereby incorporated by reference in their entireties.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the terms “N-glycan” and “glycoform” are usedinterchangeably and refer to an N-linked oligosaccharide, e.g., one thatis attached by an asparagine-N-acetylglucosamine linkage to anasparagine residue of a polypeptide. N-linked glycoproteins contain anN-acetylglucosamine residue linked to the amide nitrogen of anasparagine residue in the protein. The predominant sugars found onglycoproteins are glucose, galactose, mannose, fucose,N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialicacid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of thesugar groups occurs co-translationally in the lumen of the ER andcontinues post-translationally in the Golgi apparatus for N-linkedglycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man”refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). N-glycans differ withrespect to the number of branches (antennae) comprising peripheralsugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are addedto the Man₃GlcNAc₂ (“Man₃”) core structure which is also referred to asthe “triammnose core”, the “pentasaccharide core” or the “paucimannosecore”. N-glycans are classified according to their branched constituents(e.g., high mannose, complex or hybrid). A “high mannose” type N-glycanhas five or more mannose residues. A “complex” type N-glycan typicallyhas at least one GlcNAc attached to the 1,3 mannose arm and at least oneGlcNAc attached to the 1,6 mannose arm of a “trimannose” core. ComplexN-glycans may also have galactose (“Gal”) or N-acetylgalactosamine(“GalNAc”) residues that are optionally modified with sialic acid orderivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminicacid and “Ac” refers to acetyl). Complex N-glycans may also haveintrachain substitutions comprising “bisecting” GlcNAc and core fucose(“Fuc”). Complex N-glycans may also have multiple antennae on the“trimannose core,” often referred to as “multiple antennary glycans.” A“hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3mannose arm of the trimannose core and zero or more mannoses on the 1,6mannose arm of the trimannose core. The various N-glycans are alsoreferred to as “glycoforms.”

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, or “glycanase” or “glucosidase” which all refer to peptideN-glycosidase F (EC 3.2.2.18).

An “isolated”, “purified” or “substantially pure” nucleic acid orpolynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which issubstantially separated from other cellular components that naturallyaccompany the native polynucleotide in its natural host cell, e.g.,ribosomes, polymerases and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated”, purified or “substantially pure”also can be used in reference to recombinant or cloned DNA isolates,chemically synthesized polynucleotide analogs, or polynucleotide analogsthat are biologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence is placed adjacent to the endogenous nucleic acidsequence, such that the expression of this endogenous nucleic acidsequence is altered. In this context, a heterologous sequence is asequence that is not naturally adjacent to the endogenous nucleic acidsequence, whether or not the heterologous sequence is itself endogenous(originating from the same host cell or progeny thereof) or exogenous(originating from a different host cell or progeny thereof). By way ofexample, a promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of a hostcell, such that this gene has an altered expression pattern. This genewould now become “isolated” because it is separated from at least someof the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site and a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” in reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the genetic code, to provide an amino acid sequenceidentical to that translated from the reference nucleic acid sequence.The term “degenerate oligonucleotide” or “degenerate primer” is used tosignify an oligonucleotide capable of hybridizing with target nucleicacid sequences that are not necessarily identical in sequence but thatare homologous to one another within one or more particular segments.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990) (hereby incorporated by reference in itsentirety). For instance, percent sequence identity between nucleic acidsequences can be determined using FASTA with its default parameters (aword size of 6 and the NOPAM factor for the scoring matrix) or using Gapwith its default parameters as provided in GCG Version 6.1, hereinincorporated by reference. Alternatively, sequences can be comparedusing the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and most preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m), for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference.For purposes herein, “stringent conditions” are defined for solutionphase hybridization as aqueous hybridization (i.e., free of formamide)in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1%SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1%SDS at 65° C. for 20 minutes. It will be appreciated by the skilledworker that hybridization at 65° C. will occur at different ratesdepending on a number of factors including the length and percentidentity of the sequences which are hybridizing.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989)and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57(1988)).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked into a host cell. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Other vectors include cosmids, bacterialartificial chromosomes (BAC) and yeast artificial chromosomes (YAC).Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome (discussed in more detailbelow). Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., vectors having an origin ofreplication which functions in the host cell). Other vectors can beintegrated into the genome of a host cell upon introduction into thehost cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”).

As used herein, the term “sequence of interest” or “gene of interest”refers to a nucleic acid sequence, typically encoding a protein ofinterest or a polypeptide of interest, that is not normally expressed inthe host cell. A sequence or gene of interest includes genes andsequences that are heterologous to the host cell. Proteins andpolypeptides of interest are also often heterologous to the host cell.The methods disclosed herein allow one or more sequences of interest orgenes of interest to be stably integrated into a host cell genome.Non-limiting examples of sequences of interest include sequencesencoding one or more polypeptides of interest having an enzymaticactivity, e.g., an enzyme which affects N-glycan synthesis in a hostsuch as mannosyltransferases, N-acetylglucosaminyltransferases,UDP-N-acetylglucosamine transporters, galactosyltransferases,UDP-N-acetylgalactosyltransferase, sialyltransferases andfucosyltransferases.

The term “marker sequence” or “marker gene” refers to a nucleic acidsequence capable of expressing an activity that allows either positiveor negative selection for the presence or absence of the sequence withina host cell. For example, the P. pastoris URA5 gene is a marker genebecause its presence can be selected for by the ability of cellscontaining the gene to grow in the absence of uracil. Its presence canalso be selected against by the inability of cells containing the geneto grow in the presence of 5-FOA. Marker sequences or genes do notnecessarily need to display both positive and negative selectability.Non-limiting examples of marker sequences or genes from P. pastorisinclude ADE1, ARG4, HIS4 and URA3. For antibiotic resistance markergenes, kanamycin, neomycin, geneticin (or G418), paromomycin andhygromycin resistance genes are commonly used to allow for growth in thepresence of these antibiotics.

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which influence the expression of codingsequences to which they are operatively linked. Expression controlsequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (“expression host cell”, “expressionhost system”, “expression system” or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism. Preferred host cells are yeasts and fungi.

The term “eukaryotic” refers to a nucleated cell or organism, andincludes insect cells, plant cells, mammalian cells, animal cells andlower eukaryotic cells.

The term “lower eukaryotic cells” includes yeast, fungi,collar-flagellates, microsporidia, alveolates (e.g., dinoflagellates),stramenopiles (e.g, brown algae, protozoa), rhodophyta (e.g., redalgae), plants (e.g., green algae, plant cells, moss) and otherprotists.

The terms “yeast” and “fungi” include, but are not limited to: Pichiasp., Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichiakoclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria,Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,Saccharomyces sp., Saccharomyces cerevisiae, Hansenula polymorpha,Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillussp., Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Physcomitrella patens and Neurosporacrassa.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. On of skill in the artcan make derivatives, mutants, analogs and mimetics that mimicstructural and thus biological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally-occurring proteins and fragments thereof. A polypeptidemay be monomeric or polymeric. Further, a polypeptide may comprise anumber of different domains each of which has one or more distinctactivities. One of skill in the art can make or isolate mutants,derivatives and analogs of polypeptides.

The terms “purified” or “isolated” protein or polypeptide refers to aprotein or polypeptide that by virtue of its origin or source ofderivation (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) exists in a purity not foundin nature, where purity can be adjudged with respect to the presence ofother cellular material (e.g., is free of other proteins from the samespecies) (3) is expressed by a cell from a different species, or (4)does not occur in nature (e.g., it is a fragment of a polypeptide foundin nature or it includes amino acid analogs or derivatives not found innature or linkages other than standard peptide bonds). Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A polypeptide orprotein may also be rendered substantially free or purified of naturallyassociated components by isolation, using protein purificationtechniques well known in the art. As thus defined, “isolated” does notnecessarily require that the protein, polypeptide, peptide oroligopeptide so described has been physically removed from its nativeenvironment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide. In a preferredembodiment, the polypeptide fragment is a contiguous sequence in whichthe amino acid sequence of the fragment is identical to thecorresponding positions in the naturally-occurring sequence. Fragmentstypically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferablyat least 12, 14, 16 or 18 amino acids long, more preferably at least 20amino acids long, more preferably at least 25, 30, 35, 40 or 45, aminoacids, even more preferably at least 50 or 60 amino acids long, and evenmore preferably at least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and anti-ligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002) (herebyincorporated by reference). The term “fusion protein” refers to apolypeptide comprising a polypeptide or fragment coupled to heterologousamino acid sequences. Fusion proteins are useful because they can beconstructed to contain two or more desired functional elements from twoor more different proteins. A fusion protein comprises at least 10contiguous amino acids from a polypeptide, preferably at least 20 or 30amino acids, more preferably at least 40, 50 or 60 amino acids, andoften more preferably at least 75, 100 or 125 amino acids. Fusions thatinclude the entirety of the proteins of interest have particularutility. The heterologous polypeptide included within the fusion proteinis at least 6 amino acids in length, often at least 8 amino acids inlength, and usefully at least 15, 20, and 25 amino acids in length.Fusions also include larger polypeptides, or even entire proteins, suchas the green fluorescent protein (“GFP”) chromophore-containing proteinshaving particular utility. Fusion proteins can be produced recombinantlyby constructing a nucleic acid sequence which encodes the polypeptide ora fragment thereof in frame with a nucleic acid sequence encoding adifferent protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically bycrosslinking the polypeptide or a fragment thereof to another protein.

The term “non-peptide analog” refers to a compound with properties thatare analogous to those of a reference polypeptide. A non-peptidecompound may also be termed a “peptide mimetic” or a “peptidomimetic”.See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford UniversityPress (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: AHandbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—APractical Textbook, Springer Verlag (1993); Synthetic Peptides: A UsersGuide, (Grant, ed., W, H. Freeman and Co., 1992); Evans et al., J. Med.Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veberand Freidinger, Trends Neurosci., 8:392-396 (1985); and references sitedin each of the above, which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar to usefulpeptides of the invention may be used to produce an equivalent effectand are therefore envisioned to be part of the invention.

Amino acid substitutions can include those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2^(nd) ed.1991), which is incorporated herein by reference. Stereoisomers (e.g.,D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-, α-disubstituted amino acids, N-alkyl amino acids, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand end corresponds to the amino terminal end and the right-handend corresponds to the carboxy-terminal end, in accordance with standardusage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) In a preferred embodiment, a homologousprotein is one that exhibits at least 65% sequence homology to the wildtype protein, more preferred is at least 70% sequence homology. Evenmore preferred are homologous proteins that exhibit at least 75%, 80%,85% or 90% sequence homology to the wild type protein. In the mostpreferred embodiment, a homologous protein exhibits at least 95%, 98%,99% or 99.9% sequence identity. As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (hereinincorporated by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine L), Methionine (M),Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using a measure of homology assignedto various substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild-type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Preferred parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using PASTA, a program in GCG Version 6.1.PASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. Pearson,Methods Enzymol. 183:63-98 (1990) (herein incorporated by reference).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

As used herein, the term “comprise” or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

When referring to “mole percent” of a glycan present in a preparation ofa glycoprotein, the term means the molar percent of a particular glycanpresent in the pool of N-linked oligosaccharides released when theprotein preparation is treated with PNG'ase and then quantified by amethod that is not affected by glycoform composition, (for instance,labeling a PNG'ase released glycan pool with a fluorescent tag such as2-aminobenzamide and then separating by high performance liquidchromatography or capillary electrophoresis and then quantifying glycansby fluorescence intensity). For example, 50 mole percentGlcNAc₂Man₃GlcNAc₂Gal₂NANA₂ means that 50 percent of the releasedglycans are GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂ and the remaining 50 percent arecomprised of other N-linked oligosaccharides. In embodiments, the molepercent of a particular glycan in a preparation of glycoprotein will bebetween 20% and 100%, preferably above 25%, 30%, 35%, 40% or 45%, morepreferably above 50%, 55%, 60%, 65% or 70% and most preferably above75%, 80% 85%, 90% or 95%.

As used herein, the term “predominantly” or variations such as “thepredominant” or “which is predominant” will be understood to mean theglycan species that has the highest mole percent (%) of total N-glycansafter the glycoprotein has been treated with PNGase and released glycansanalyzed by mass spectroscopy, for example, MALDI-TOF MS. In otherwords, the phrase “predominantly” is defined as an individual entity,such as a specific glycoform, is present in greater mole percent thanany other individual entity. For example, if a composition consists ofspecies A in 40 mole percent, species B in 35 mole percent and species Cin 25 mole percent, the composition comprises predominantly species A.

The term “therapeutically effective amount” refers to an amount of therecombinant erythropoietin of the invention which gives an increase inhematocrit that provides benefit to a patient. The amount will vary fromone individual to another and will depend upon a number of factors,including the overall physical condition of the patient and theunderlying cause of anemia. For example, a therapeutically effectiveamount of erythropoietin of the present invention for a patientsuffering from chronic renal failure can be in the range of 20 to 300units/kg or 0.5 ug/kg to 500 ug/kg based on therapeutic indication. Theterm “unit” refers to units commonly known in the art for assessing theactivity of erythropoietin compositions. A milligram of pureerythropoietin is approximately equivalent to 150,000 units. A dosingschedule can be from about three times per week to about once every fouror six weeks. The actual schedule will depend on a number of factorsincluding the type of erythropoietin administered to a patient (EPO orPEGylated-EPO) and the response of the individual patient. The higherdose ranges are not typically used in anemia applications but can beuseful on other therapeutic applications. The means of achieving andestablishing an appropriate dose of erythropoietin for a patient is wellknown and commonly practiced in the art.

Variations in the amount given and dosing schedule from patient topatient are including by reference to the term “about” in conjunctionwith an amount or schedule. The amount of erythropoietin used fortherapy gives an acceptable rate of hematocrit increase and maintainsthe hematocrit at a beneficial level (for example, usually at leastabout 30% and typically in a range of 30% to 36%). A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one skilled in the art using publicly available materials andprocedures. Additionally, iron may be given to the patient to maintainincreased erythropoiesis during therapy. The amount to be given may bereadily determined by methods commonly used by those skilled in the art.

The erythropoietin of the present invention may thus be used tostimulate red blood cell production and correct depressed red celllevels. The most common therapeutic application of erythropoietin is tocorrect red cell levels that are decreased due to anemia. Among theconditions treatable by the present invention include anemia associatedwith a decline or loss of kidney function (chronic renal failure),anemia associated with myelosuppressive therapy, such aschemotherapeutic or anti-viral drugs (such as AZT), anemia associatedwith the progression of non-myeloid cancers, and anemia associated withviral infections (such as HIV). Additionally, erythropoietin of thepresent invention can be used to prevent or lessen neuronal damagefollowing stroke (particularly a non-sialylated epo), congestive heartfailure, in the treatment of spinal injury; i.e., anti-inflammatory,anti-apoptosis and the recruitment of stem cells apart fromerythropoiesis. Also treatable are conditions which may lead to anemiain an otherwise healthy individual, such as an anticipated loss of bloodduring surgery. In general, any condition treatable with erythropoietinsgenerally can be treated with the erythropoietins of the presentinvention.

I. Glycosylation

The invention provides methods and materials for the transformation,expression and selection of recombinant proteins, particularlyerythropoietin, in lower eukaryotic host cells, which have beengenetically engineered to produce glycoproteins with specific desiredN-glycans as the predominant species. In certain embodiments, theeukaryotic host cells have been genetically engineered to produceerythropoietin, or a variant of erythropoietin, with a specific desiredN-glycan as the predominant species. In preferred embodiments, thepredominant N-glycan is one which is not immunogenic to mammals,particularly humans, or which has reduced immunogenicity compared tothat of hypermannosylated glycoproteins. Exemplary glycosylationpatterns are shown in FIGS. 9A-9B.

In a specific preferred embodiment, the predominant N-glycan is a fullysialylated glycan, represented as: GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂. In otherpreferred embodiments, the predominant N-glycan may be partiallysialylated or asialylated glycan; fully galactosylated; partiallygalactosylated; or agalactosylated. Thus, preferred embodiments includethose in which the predominant N-glycan is GlcNAc₂Man₃GlcNAc₂Gal₂NANA;GlcNAc₂Man₃GlcNAc₂GalNANA; GlcNAc₂Man₃GlcNAc₂Gal₂;GlcNAc₂Man₃GlcNAc₂Gal, GlcNAc₂Man₃GlcNAcGal; GlcNAc₂Man₃GlcNAc₂,GlcNAc₂Man₃GlcNAcr or GlcNAc₂Man₃. In other preferred embodiments, thepredominant N-glycan is a partially galactosylated glycans, representedas GlcNAc₂Man₃GlcNAc₂Gal_(n), where n may be 1 or 2.

In other embodiments of the present invention, the predominant N-glycanmay be a hybrid glycoform, represented by the formula:GlcNAc₂Man₍₄₋₅₎GlcNAc₍₀₋₁₎Gal₍₀₋₁₎NANA₍₀₋₁₎. Preferred embodimentsinclude those in which the predominant N-glycan isGlcNAc₂Man₅GlcNAcGalNANA, GlcNAc₂Man₅GlcNAcGal; and GlcNAc₂Man₅GlcNAcand GlcNAc₂Man₅.

Many wild-type lower eukaryotic cells, including yeasts and fungi, suchas Pichia pastoris, produce glycoproteins without any core fucose. Thus,in the above embodiments, the recombinant glycoproteins produced inaccordance with the present invention may lack fucose, or be essentiallyfree of fucose. Alternatively, in certain embodiments, the recombinantlower eukaryotic host cells may be genetically modified to include afucosylation pathway, thus resulting in the production of recombinantglycoprotein compositions in which the predominant N-glycan species isfucosylated. Unless specifically noted, the glycoprotein compositions ofthe present invention may be produced either in afucosylated form, orwith core fucosylation present.

In the present invention, the recombinant glycoprotein produced inaccordance with the above description is then chemically modified toimprove its physical characteristics, notably serum half-life andpharmacokinetics. In preferred embodiments, the chemical modification isaccomplished by linking one or more polyethylene glycol (PEG) moietiesto said recombinant glycoprotein, resulting in a PEGylated glycoprotein.In certain preferred embodiments, the recombinant glycoprotein ismodified by linking one or more PEG moieties to the N-terminal aminoacid of the recombinant glycoprotein, resulting in an N-terminallyPEGylated glycoprotein.

II Erythropoietin

Erythropoietin (EPO) is a haemopoietic glycoprotein produced in thekidney that stimulates the differentiation of late erythroid progenitorcells to mature red blood cells. Erythropoietin exerts its biologicalactivity by binding to receptors on erythroid precursors. Theerythropoietin gene is not particular to humans. Analogous genes havebeen found in other species, including many mammals (See Wen et al.,Blood (1993); 82:1507-16). Therefore, both human and non-human genes canbe expressed as described herein and the variety of erythropoietins mayalso be useful in the methods of the present invention.

Naturally occurring human erythropoietin is first translated to a 166amino acid containing polypeptide chain with arginine at position 166.In a postranslational modification, arginine 166 is cleaved by acarboxypeptidase. The primary structure of 165 amino acid humanerythropoietin is shown in SEQ ID NO:2 and a nucleic acid this EPO isshown in SEQ ID NO:1. The secondary structure of erythropoietin includestwo disulfide bridges between Cys7-Cys161 and Cys29-Cys33. Fullyglycosylated EPO comprises approximately 40% carbohydrate groups bymolecular weight (Sasaki, H., et al., J. Biol. Chem. 262 (1987)12059-12076). The molecular weight of the polypeptide chain of humanerythropoietin without the glycan moieties is 18,236 Da.

Because erythropoietin is essential in red blood cell formation, it isuseful in the treatment of blood disorders characterized by low ordefective red blood cell production. Clinically, erythropoietin is usedin the treatment of various ailments, for example, anemia in chronicrenal failure patients (CRF) and in AIDS and cancer patients undergoingchemotherapy (Danna, R. P., et al., In: M B, Garnick, ed. Erythropoietinin Clinical Applications—An International Perspective. New York, N.Y.:Marcel Dekker; 1990, pp. 301-324). However, the bioavailability ofcurrently available protein therapeutics such as erythropoietin islimited by their short plasma half-life and susceptibility to proteasedegradation. These shortcomings prevent them from attaining maximumclinical potency. Modifications of the amino acid sequence of EPO havebeen disclosed, for example, in a number of references including U.S.Pat. No. 4,835,260; WO 94/25055; WO 94/24160; WO 94/02611; WO 95/05465.

Erythropoietin has been manufactured biosynthetically using recombinantDNA technology (Egrie, J. C., et al., Immunobiol. 72 (1986) 213-224).Erythropoietin currently used in human therapy is the product of acloned human EPO gene inserted into and expressed in the ovarian tissuecells of the Chinese hamster (CHO cells). Both human urinary derivederythropoietin and recombinant erythropoietin (expressed in mammaliancells) contain three N-linked and one O-linked oligosaccharide chainswhich together comprise about 40% of the total molecular weight of theglycoprotein. N-linked glycosylation occurs at asparagine residueslocated at positions 24, 38 and 83 while O-linked glycosylation occursat a serine residue located at position 126 (Lai, et al., J. Biol. Chem.261 (1986) 3116; Broudy, V. C., et al., Arch. Biochem. Biophys. 265(1988) 329). The oligosaccharide chains have been shown to be modifiedwith terminal sialic acid residues. Enzymatic treatment of glycosylatederythropoietin to remove all sialic acid residues results in a loss ofin vivo activity but does not affect in vitro activity (Lowy et al.,Nature 185 (1960) 102; Goldwasser, E., et al. J. Biol. Chem. 249 (1974)4202-4206). This behavior has been explained by rapid clearance ofasialoerythropoietin from circulation upon interaction with the hepaticasialoglycoprotein binding protein (Morrell et al., J. Biol. Chem. 243(1968) 155; Briggs, D. W., et al., Am. J. Physiol. 227 (1974) 1385-1388;Ashwell, G., and Kawasaki, T., Methods Enzymol. 50 (1978) 287-288).Thus, erythropoietin possesses in vivo biological activity only when itis sialylated to avoid its binding by the asialoglycoprotein bindingprotein.

The role of the other components in the oligosaccharide chains oferythropoietin has not been well defined. It has been shown thatpartially diglycosylated erythropoietin has greatly reduced in vivoactivity compared to the glycosylated form but does retain in vitroactivity. (Dordal, M. S., et al., Endocrinology 116 (1985) 2293-2299).In another study, however, the removal of N-linked or O-linkedoligosaccharide chains singly or together by mutagenesis of asparagineor serine residues that are glycosylation sites sharply reducesbiological activity of the altered erythropoietin that is produced inmammalian cells (Dube, S., et al., J. Biol. Chem. 263 (1988)1751647521).

Oligonucleotide-directed mutagenesis has been used to prepare structuralmutants of EPO lacking specific sites for glycosylation (Yamaguchi, K.,et al., J. Biol. Chem. 266 (1991) 20434-20439; and Higuchi, M., et al.,J. Biol. Chem. 267 (1992) 7703-7709). Cloning and expression ofnon-glycosylated EPO in E. coli is described by Lee-Huang, S., Proc.Natl. Acad. Sci. USA 61 (1984) 2708-2712; and in U.S. Pat. No.5,641,663.

EP 0 640 619 relates to analogs of human erythropoietin comprising anamino acid sequence which includes at least one additional site forglycosylation. The added sites for glycosylation may result in a greaternumber of carbohydrate chains, and higher sialic acid content, thanhuman erythropoietin. Erythropoietin analogs comprising amino acidsequences which include the rearrangement of at least one site forglycosylation are also provided. Analogs comprising an addition of oneor more amino acids to the carboxy terminal end of erythropoietinwherein the addition provides at least one glycosylation site are alsoincluded.

Compared to erythropoietin produced in CHO cells, erythropoietinproduced in Pichia with humanized glycosylation is much more uniform inthe attached oligosaccharide structures that comprise N- andO-glycosylation. CHO-derived rhEPO is produced with a mixture ofglycoforms, including bi-, tri- and tetra-antennary forms with varyingamounts of sialylation. Process development is used to enrich fortetra-antennary sialylated glycoforms which comprise a small portion ofthe of N-glycans pre-enrichment (Restelli et al, 2006 Biotechnology andBioengineering 94 (3) p. 481-494). Additionally, while sialylation inhumanized yeast does not include N-glycolylneuraminic acid (NGNA),sialyated erythropoietin produced in CHO cells contains a mixture ofNANA (N-acetylneuraminic acid) and NGNA.

Erythropoietins produced in mammalian cell lines, such as Chinesehamster ovary (CHO) cells are enriched for carbohydrates that containsialylated N-linked glycans. Enriching for tetra-antennary glycoformsincreases the proportion of lactosamine repeats on N-glycans oferythropoietin. Erythropoietin molecules containing these repeats maypossess impaired in vivo efficacy as presence of polylactosamine repeatshas been correlated with rapid clearance from the circulation via theliver (Fukuta et al., (1989) Blood, Vol. 73, 84).

Lactosamine moieties have also been reported to bind to the galectinfamily of lectins, carbohydrate binding proteins differentiallyexpressed on the cell surface of different cell and tissues types.Specifically, galectin-3 specifically recognizes lactosamine. Galectin-3has been found to be overexpressed on the cell surface of many differenttumor cell types and has been implicated in cell growth, transformationand metastasis (Deininger et al., (2002) Anticancer Research, Vol. 22,1585).

Thus, glycoproteins that contain lactosamine repeats can potentially betargeted to cells expressing galectin-3 on the cell surface. Moreover,this presents a further potential risk that lactosamine-containingglycoproteins may selectively target tumor cells which maycoincidentally bear the cognate receptor for the glycoprotein. In thecase of erythropoietins, the fraction of erythropoietin that containslactosamine may preferentially target tumor cells and, if theerythropoietin receptor is present, an aberrant mitogenic signal mayarise driving tumor cell growth with metastatic potential.

Erythropoietins produced in Pichia pastoris lack undesired glycoformssuch as lactosamine repeats. This would likely alleviate concernsrelating to the tumorigenic potential of EPO and other glycoproteins.

Other differences include a complete lack of attached core fucose andpolylactosamine in Pichia-produced erythropoietin, both present on theCHO cell version, and variations on O-glycan composition, with aheterogeneous mixture of O-GalNAc structures on CHO-producederythropoietin compared to the O-mannose that may be found onPichia-produced erythropoietin. Sialic acid linkage is primarily α2,3 inCHO-produced erythropoietin, with some α2,6 present, whilePichia-produced erythropoietin contains exclusively α2,6 (Hamilton,Science Vol 313, p. 1441-1443), similar to human urinary erythropoietinwhich contains predominantly α2,6 linked sialic acid.

Erythropoietin (EPO) is a tissue-protective cytokine that has been shownto prevent vascular spasm, apoptosis, and inflammatory responses.Although best known for its activity on hematopoiesis, EPO also affectsother tissues, including the nervous system. Animal models havedemonstrated that single doses of rhEPO are efficacious for thetreatment of acute injury (4-6, 19). For instance, infusion of EPO intothe lateral ventricle of gerbils subjected to occlusion of the commoncarotid arteries prevented ischemia-induced learning disability andrescued hippocampal neurons from degeneration (Bernaudin et al. (1999) JCereb Blood Flow Metab 19, 643).

Studies in vivo have demonstrated the protective effects of EPO onvarious forms of neuronal damage (Brines et al. (2000), PNAS, 97, 10526;Celik et al. (2002), PNAS, 99, 2258; Junk et al. (2002), PNAS, 99,10659). However, many clinical situations will likely require multipledoses of rhEPO, which most likely will lead to potentially harmfulincreases in hematocrit thus curbing enthusiasm for recombinant humanerythropoietin (rhEPO) as a potential neuroprotective therapeutic. Thisis supported by animal models which clearly show that EPO-dependentincreases in hematocrit can cause and amplify brain injury. A potentialsolution to this paradox could be through the use of EPO withbi-antennary glycosylation which has a minimal effect on hematocrit inanimal models (Hamilton et al. (2006) Science 313, 1441). Thus multiplehigh doses of EPO with biantennary glycosylation for the treatment ofinflammation may have little effect on overall hematocrit.

Another interesting glycoform that can be used for the treatment ofinflammation is a bi-antennary EPO without terminal sialylation. Thismay also serve as an effective treatment as an anti-inflammatory agentwithout significantly increasing hematocrit. The added advantage forthis molecule would be its reduced affinity for the asialo-glycoproteinreceptor in the liver relative to the preferred substrates of thisreceptor which are the tri- and tetra-antennary terminallygalactosylated glycoforms. Therefore the advantages of an EPO withbiantennary asialylated glycosylation are reduced liver clearance,little effect on hematocrit and preffered neuronal tissue distribution.Asialo-EPO can be made according to this invention by expressing EPO inyeast cells lacking the sialyation portion of the glycosylation pathwayshown herein.

III. Nucleic Acid Encoding the Glycoprotein

The erythropoietins of the present invention are encoded by nucleicacids. The nucleic acids can be DNA or RNA, typically DNA. The nucleicacid encoding the glycoprotein is operably linked to regulatorysequences that allow expression of the glycoprotein. Such regulatorysequences include a promoter and optionally an enhancer upstream, or 5′,to the nucleic acid encoding the fusion protein and a transcriptiontermination site 3′ or down stream from the nucleic acid encoding theglycoprotein. The nucleic acid also typically encodes a 5′ UTR regionhaving a ribosome binding site and a 3′ untranslated region. The nucleicacid is often a component of a vector which transfers to nucleic acidinto host cells in which the glycoprotein is expressed. The vector canalso contain a marker to allow recognition of transformed cells.However, some host cell types, particularly yeast, can be successfullytransformed with a nucleic acid lacking extraneous vector sequences.

Nucleic acids encoding desired erythropoietin of the present inventioncan be obtained from several sources. cDNA sequences can be amplifiedfrom cell lines known to express the glycoprotein using primers toconserved regions (see, e.g., Marks et al., J. Mol. Biol. 581-596(1991)). Nucleic acids can also be synthesized de novo based onsequences in the scientific literature. Nucleic acids can also besynthesized by extension of overlapping oligonucleotides spanning adesired sequence of a larger nucleic acid, e.g., genomic DNA (see, e.g.,Caldas et al., Protein Engineering, 13, 353-360 (2000)).

The present invention preferably employs a nucleic acid encoding amammalian erythropoietin and most preferably employs a nucleic acidencoding a human erythropoietin. Human erythropoietin is well known inthe art as a 165 or 166 amino acid protein.

The following nucleotide and amino acid sequences are exemplarypreferred sequences that can be employed in present invention.

EPO nucleotide sequence [SEQ ID NO: 1]ATGAGATTTC CTTCAATTTT TACTGCTGTT TTATTCGCAGCATCCTCCGC ATTAGCTGCT CCACCAAGAT TGATTTGTGACTCCAGAGTT TTGGAGAGAT ACTTGTTGGA GGCTAAAGAGGCTGAGAACA TCACTACTGG TTGTGCTGAA CACTGTTCCTTGAACGAGAA CATCACAGTT CCAGACACTA AGGTTAACTTCTACGCTTGG AAGAGAATGG AAGTTGGACA ACAGGCTGTTGAAGTTTGGC AAGGATTGGC TTTGTTGTCC GAGGCTGTTTTGAGAGGTCA AGCTTTGTTG GTTAACTCCT CCCAACCATGGGAACCATTG CAATTGCACG TTGACAAGGC TGTTTCTGGATTGAGATCCT TGACTACTTT GTTGAGAGCT TTGGGTGCTCAGAAAGAGGC TATTTCTCCA CCAGATGCTG CTTCAGCTGCTCCATTGAGA ACTATCACTG CTGACACTTT CAGAAAGTTGTTCAGAGTTT ACTCCAACTT CTTGAGAGGA AAGTTGAAGTTGTACACTGG TGAAGCTTGT AGAACTGGTG ACTAGTAA EPO Amino Acid sequence[SEQ ID NO: 2] APPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENITVPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQALLVNSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAISPPDAASAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDIV. Host Cells

Lower eukaryotic cells, such as yeast and fungi, are preferred forexpression of the erythropoietin of the present invention because theycan be economically cultured, give high yields, and when appropriatelymodified are capable of suitable glycosylation. Yeast particularlyoffers established genetics allowing for rapid transformations, testedprotein localization strategies and facile gene knock-out techniques.Suitable vectors have expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences and the like as desired.

Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica,and Hansenula polymorphs are preferred for cell culture because they areable to grow to high cell densities and secrete large quantities ofrecombinant protein. Likewise, filamentous fungi, such as Trichodermareesei, Aspergillus niger, Fusarium sp, Neurospora crassa and others canbe used to produce glycoproteins of the invention.

Lower eukaryotes, particularly yeast and fungi, can be geneticallymodified so that they express glycoproteins in which the glycosylationpattern is human-like or humanized. This can be achieved by eliminatingselected endogenous glycosylation enzymes and/or supplying exogenousenzymes as described by Gerngross et al., US 20040018590 and U.S. Pat.No. 7,029,872, the disclosures of which are hereby incorporated hereinby reference. For example, a host cell can be selected or engineered tobe depleted in 1,6-mannosyl transferase activities, which wouldotherwise add mannose residues onto the N-glycan on a glycoprotein.

Using the methods and materials of the present invention, it is possibleto produce glycoprotein compositions comprising a plurality ofglycoforms, each glycoform comprising at least one N-glycan attachedthereto, wherein the glycoprotein composition thereby comprises aplurality of N-glycans in which a predominant glycoform comprises adesired N-glycan. Utilizing the tools described in Gerngross et al., US20040018590 and U.S. Pat. No. 7,029,872, together with the presentinvention, it is possible to produce many different N-linked glycoforms.Depending upon the specific needs, the methods of the present inventioncan be used to obtain glycoprotein composition in which the predominantN-glycoform is present in an amount between 5 and 80 mole percentgreater than the next most predominant N-glycoform; in preferredembodiments, the predominant N-glycoform may be present in an amountbetween 10 and 40 mole percent; 20 and 50 mole percent; 30 and 60 molepercent; 40 and 70 mole percent; 50 and 80 mole percent greater than thenext most predominant N-glycoform. In other preferred embodiments, thepredominant N-glycoform is a desired N-glycoform and is present in anamount of greater than 25 mole percent; greater than 35 mole percent;greater than 50 mole percent; greater than 60 mole percent; greater than75 mole percent; or greater than 80 mole percent of the total number ofN-glycans.

In preferred embodiments, a vector can be constructed with one or moreselectable marker gene(s), and one or more desired genes encodingerythropoietin which is to be transformed into an appropriate host cell.For example, one or more genes selectable marker gene(s) can bephysically linked with one or more gene(s), expressing a desirederythropoietin peptide or protein for isolation or a fragment of saiderythropoietin peptide or protein having the desired activity can beassociated with the selectable gene(s) within the vector. The selectablemarker gene(s) and erythropoietin gene(s) can be arranged on one or moretransformation vectors so that presence of the erythropoietin gene(s) ina transformed host cell is correlated with expression of the selectablemarker gene(s) in the transformed cells. For example, the two genes canbe inserted into the same physical plasmid, under control of a singlepromoter, or under the control of two separate promoters. It may also bedesired to insert the genes into distinct plasmids and co-transformedinto the cells.

Other cells useful as host cells in the present invention includeprokaryotic cells, such as E. coli, and eukaryotic host cells in cellculture, including mammalian cells, such as Chinese Hamster Ovary (CHO).

V. Chemically Modified Erythropoietin

As noted above, polymer vehicles may be conjugated to proteins such aserythropoietin in order to enhance the properties. Various means forattaching chemical moieties useful as vehicles are currently available,see, e.g., Patent Cooperation Treaty (“PCT”) International PublicationNo. WO 96/11953, entitled “N-Terminally Chemically Modified ProteinCompositions and Methods,” herein incorporated by reference in itsentirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

Chemically modified erythropoietin compositions (i.e., “derivatives”),where the protein or polypeptide is linked to a polymer, are includedwithin the scope of the present invention. The polymer selected istypically water soluble so that the protein to which it is attached doesnot precipitate in an aqueous environment, such as a physiologicalenvironment. The polymer selected is usually modified to have a singlereactive group, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled asprovided for in the present methods. Included within the scope ofmodified erythropoietin compositions is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol), andpolyvinyl alcohol. For the acylation reactions, the polymer(s) selectedshould have a single reactive ester group. For reductive alkylation, thepolymer(s) selected should have a single reactive aldehyde group. Thepolymer may be of any molecular weight, and may be branched orunbranched. A particularly preferred water-soluble polymer for useherein is polyethylene glycol, abbreviated PEG. As used herein,polyethylene glycol is meant to encompass any of the forms of PEG thathave been used to derivatize other proteins, such as mono-(C1-C10)alkoxy or aryloxy-polyethylene glycol.

PEGylation (i.e. modification by the addition of PEG or a PEGderivative), of PAL may be carried out by any of the PEGylationreactions known in the art, as described for example in the followingreferences: Focus on Growth Factors 3:4-10 (1992); EP 0 154 316; and EP0 401 384. Preferably, the PEGylation is carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer), as describedbelow.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparingPEGylated-erythropoietin will generally comprise the steps of (a)reacting an erythropoietin polypeptide with polyethylene glycol (such asa reactive ester or aldehyde derivative of PEG), under conditionswhereby erythropoietin becomes attached to one or more PEG groups, and(b) obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-PEGylated product.

The present invention also provides a method employing reductivealkylation. for selectively obtaining N-terminally chemically modifiederythropoietin. In this method linkage to the N-terminus oferythropoietin can be targeted because of the differential reactivity ofdifferent primary amino groups. One chooses a pH wherein the pKa betweenthe ε-amino group of lysines in the protein and the α-amino group of theN-terminus results in nearly selective derivatization of the protein atthe N-terminus by a reaction with a carbonyl group or PEG or anotherpolymer. Mono-polymer modified erythropoietin is preferred. Thepreparations of this invention will preferably be greater than 50%, 55%,60%, 65%, 70% or 75% mono-polymer erythropoietin, more preferablygreater than 80%, 85% or 90% mono-polymer:protein conjugate, and mostpreferably greater than 95% mono-polymer-erythropoietin conjugate.

The method of obtaining the mono-polymer derivatized erythropoietinpreparation may be by purification of the derivatized material from apopulation of non-derivatized erythropoietin molecules after theconjugation. For example, presented below is an example wheremono-PEGylated erythropoietin is separated using chromatography. Sizeexclusion chromatography, ion exchange chromatography or a combinationof the two and potentially other common purification methods can beused. These methods can be used as analytical tools to characterize thepurified products or as a preparative purification tools.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be linear or branched.The average molecular weight of the PEG will preferably range from about2 kiloDalton (“kDa”) to about 100 kDa, more preferably from about 5 kDato about 60 kDa, more preferably from about 20 kDa to about 50 kDa; mostpreferably from about 30 kDa to about 40 kDa. These PEGs can be suppliedfrom any commericial vendors including NOF Corporation (Tokyo, Japan),Dow Pharma (ChiroTech Technology, Cambridge, UK), Nektar (San Carlos,Calif.) and SunBio (Anyang City, South Korea). Suitable PEG moietiesinclude, for example, 40 kDa methoxy poly(ethylene glycol)propionaldehyde; 60 kDa methoxy poly(ethylene glycol) propionaldehyde;31 kDa alpha-methyl-w-(3-oxopropoxy), polyoxyethylene; 30 kDa PEG: 30kDa Methoxy poly(ethylene glycol) propionaldehyde and 45 kDa2,3-Bis(methylpolyoxyethylene-oxy)-1-[(3-oxopropyl)polyoxyethylene-oxy]-propane. The PEG groups will generally be attachedto the compounds of the invention via acylation or reductive aminationthrough a reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the protein or polypeptideof interest (e.g., an aldehyde, amino, or ester group). For example, thePEG moiety may be linked to the N-terminal amino acid residue oferythropoietin, either directly or through a linker.

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis (see, for example, FIGS. 5 and 6 andthe accompanying text herein). The peptides are “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulins has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference. Dextran of about 1 kD to about 20 kD is preferred whendextran is used as a vehicle in accordance with the present invention.

As described above, the presence of a “linker” group is optional. Whenpresent, its chemical structure is not critical, since it servesprimarily as a spacer. The linker is preferably made up of amino acidslinked together by peptide bonds. Thus, in preferred embodiments, thelinker is made up of from 1 to 20 amino acids linked by peptide bonds,wherein the amino acids are selected from the 20 naturally occurringamino acids. Some of these amino acids may be glycosylated, as is wellunderstood by those in the art. In a more preferred embodiment, the 1 to20 amino acids are selected from glycine, alanine, proline, asparagine,glutamine, and lysine. Even more preferably, a linker is made up of amajority of amino acids that are sterically unhindered, such as glycineand alanine. Thus, preferred linkers are polyglycines (particularly(Gly)₄, (Gly)₅, poly(Gly-Ala), and polyalanines. Other specific examplesof linkers are:

(Gly)₃Lys(Gly)₄;

(Gly)₃AsnGlySer(Gly)₂;

(Gly)₃Cys(Gly)₄; and

GlyProAsnGlyGly.

To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are alsopreferred. The linkers shown here are exemplary; linkers within thescope of this invention may be much longer and may include otherresidues.

Non-peptide linkers are also possible. For example, alkyl linkers suchas—NH—(CH₂)s-C(O)—, wherein s=2-20 could be used. These alkyl linkersmay further be substituted by any non-sterically hindering group such aslower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2,phenyl, etc. An exemplary non-peptide linker is a PEG linker, wherein nis such that the linker has a molecular weight of 100 to 5000 kD,preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

PEGylation of glycosylated EPO is described in WO 01/02017. Suchmolecules show an improved biological activity. WO00/32772 and Francis,G. E., et al., Int. J. Hem. 68 (1988) 1-18, describe polyethyleneglycol-modified non-glycosylated EPO. The molecules of WO 00/32772 areadditionally modified at positions 166. Such molecules are described asnot causing a significant increase in hematocrit. The PEG-polymerportion consists of 1-5 polymer chains. WO 00/32772 suggests to controlthe degree and site of PEGylation by lowering the pH and reducing thePEG:amine ratio. Reactions run at pH 7 and 1.5:1 molar ratio ofPEG-aldehyde: amine groups, preferentially react with the N-terminalα-amino group.

Some useful PEGylation linkages are shown in Table 1 and some usefulPEGylation reactions are shown in FIG. 12. In a preferred method,N-terminal PEGylation is accomplished through the use ofmPEG-propionaldehyde and its covalent conjugation to the rhEPON-terminus via reductive amination. Selectivity is achieved byexploiting the difference in pKa values between the ε-amino group oflysine (pKa˜10) and the N-terminal amino group (pKa˜7.6-8.0). In atypical derivatization reaction 30% to 50% of the rhEPO is PEGylated.Higher degrees of derivatization may be achieved under optimizedconditions. Mono-PEGylated rhEPO is then purified using a cationexchange chromatography step. Preferably, the purified mono-PEGylatedrhEPO is greater than 80%, 85%, 90% or most preferably greater than 95%of the rhEPO after chromatography.

TABLE 1 PEGylation: Reagent selection, PEG-conjugate linkage andconjugate stability Conjugate Linkage Formation Amine PEGylationmPEG-p-nitrophenyl carbonate Carbamate mPEG-propionaldehyde and areducing agent Secondary amine (stable) mPEG-NHS esters Amide ThiolPEGylation mPEG-Maleimide Thioether Carboxyl PEGylation mPEG-amine and acoupling agent Amide (stable)

Generally, conditions which may be alleviated or modulated byadministration of the present polymer/erythropoietin-derivatives includethose described herein for erythropoietin molecules in general. However,the polymer/erythropoietin and erythropoietin-derivative moleculesdisclosed herein may have additional activities, enhanced or reducedactivities, or other characteristics, as compared to the non-derivatizedmolecules

EXAMPLES Example 1 Construction of the Genetically Engineered Pichia 6.0Cell Line

Following the procedures disclosed in Gerngross U.S. Pat. No. 7,029,872and Gerngross US 20040018590, one can construct vectors that are usefulfor genetically engineering lower eukaryotic host cells such that theyare capable of expressing a desired polypeptide having a desiredN-glycoform as the predominant species. Beginning with the wild-typestrain of Pichia pastoris NRRL-11430, stepwise introduction of genes ismade according to the series described in FIG. 1. The genotype of strainYGLY3159 used herein is ura5Δ::MET16 och1 Δ::lacZ bmt2Δ::lacZ/KlMNN2-2,mnn4L1Δ::lacZ/MmSLC35Δ3 Δpno1Δmnn4Δ::lacZ met16Δ::lacZ,his1Δ::lacZ/ScGAL10/XB33/DmUGT, arg1Δ::HIS1/KD53/TC54,ADE1::lacZ/NA10/MmSLC35A3/FB8, PRO1::lacZ-URA5-lacZ/TrMDS1, AOX1:Shble/AOX1p/ScαMFpre-GFI800, TRP2::ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33.

The plasmids used for construction of the strain YGLY3159 areillustrated in FIG. 2, Panels A through O. The plasmids are transformedinto the desired cell, in accordance with standard techniques. Suitabletechniques for the construction of cell lines are also demonstrated inHamilton et al., Science 313:1441:1443 (2006), the disclosure of whichis hereby incorporated herein by reference.

Example 2 Construction of the Vector for Production of Recombinant EPO

Oligos to the alpha mating factor presequence from Saccharomycescerevisiae were phosphorylated and annealed to create an EcoRI overhangat the 5′ end and a blunt end at the 3′ end. This oligo pair was thenligated to the coding DNA sequence encoding human erythropoietin, asshown in FIG. 2, to form pGLY2088, which was transformed into Pichiapastoris as shown in FIG. 1.

Example 3 Transformation and Fermentation of the 6.0 Cell Line

A. Transformation

Yeast strains were transformed by electroporation (using standardtechniques as recommended by the manufacturer of the electroporatorBioRad).

B. Fermentation Process Description

Fermentation runs were carried out in 15 L (12 L working volume)autoclavable glass bioreactors from Applikon. The reactor is inoculated(0.04% v/v) with an exponential phase shake flask culture grown from afrozen stock vial. The batch phase ends in 24-36 hours upon depletion ofthe initial charge glycerol. The wet cell weight (WCW) after the batchphase is typically 120±25 g/L WCW. At this point a 50% w/w glycerolsolution containing 12 mL/L PTM1 salts is fed to the fermenter in asingle pulse leading to a final glycerol concentration of 30 g/L at thestart of the glycerol fed-batch phase. A solution containing a syntheticinhibitor of fungal O-glycosylation (PMTi-3) dissolved in methanol at2.6 mg/mL is added at 1 mL/L. A protease inhibitor cocktail (45 mg/mLPepstatin A and 15 mM of Chymostatin in DMSO) is added at 0.6 mL/L.Within 4 hours the glycerol is consumed and the wet cell weight hasreached 225±25 g/L WCW. Gene expression is then induced by theinitiation of a methanol feed containing 12 mL/L of PMT1 salts at 2.3g/h/L. At the start of the methanol feed batch phase as well as every 24hours of induction, 1 mL/L of 2.6 mg/mL PMTi-3 in methanol and 0.6 mL/Lof the protease inhibitor cocktail are added. Induction continues for 40hours when the final wet cell weight is expected to be approximately300±25 g/L. (L* is the initial charge volume before inoculation).

Primary clarification of fermentor broth is performed by centrifugation.The whole cell broth is transferred into 1000 mL centrifuge bottles andcentrifuged at 4° C. for 15 minutes at 13,000×g.

Example 4 Purification

Human EPO (hEPO) for PEGylation was generated by a three-stepchromatographic separation, as follows, in which 95% purity was achieved(FIG. 3). First, cell-free fermentation supernatant was filtered through0.2 μm membrane filter, concentrated and buffer exchanged using aMiniKross tangential flow separation module with hollow fiber membrane.

Q sepharose Big Beads were used in the first step to capture the hostcell proteins and hEPO flowthrough. The pool of flowthrough and the washsamples from the Q sepharose Big Beads column were adjusted to pH 5.0with acetic acid. Conductivity was measured to ensure a value ofapproximately 4.5 mS/cm.

Next, the sample was filtered through a 0.2 μm membrane filter andloaded on to a SP sepharose Fast Flow column pre-equilibrated with threecolumn volumes of 50 mM sodium acetate pH 5.0. Ten column volumes of agradient from 50 mM sodium acetate pH 5.0 to 20 mM sodium acetate pH5.0; 500 mM NaCl was applied to elute the protein, followed by a stepelution with 0.5 column volumes 20 mM sodium acetate pH 5.0; 750 mMNaCl. Fractions containing hEPO were pooled and dialyzed in 50 mM TRISpH 7.0.

The hEPO containing fractions were loaded on to a Blue Sepharose 6 FFcolumn pre-equilibrated with three column volumes of 50 mM TRIS pH 7.0,Ten column volumes of a linear gradient from 50 mM TRIS pH 7.0 to 50 mMTRIS pH 8; 3M NaCl were applied, followed by a step elution of twocolumn volumes with 50 mM TRIS pH 8; 3M NaCl.

The fractions that displayed an EPO band (average molecular weight ˜25kDa) were pooled, filtered through a 0.2 μm membrane filter, dialyzed in20 mm MES pH 6.0 at 4° C. and stored at 4° C. The sample pool was thenconcentrated and dialyzed in 50 mM sodium acetate buffer at pH 5.2 to aprotein concentration of 1 mg/ml.

It has been seen that exposure of fermentation supernatant to pH 5increased the loss of hEPO. It is therefore important to maintain thecapture and intermediate step of hEPO purification at neutral pH.

An overview of a second general purification scheme is shown in FIG. 10.A purified sample prepared using this scheme was analyzed by SDS-PAGEshown in FIG. 11.

Primary clarification is performed by centrifugation. The whole cellbroth is transferred into 1000 mL centrifuge bottles and centrifuged at4° C. for 15 minutes at 13,000×g. An ultrafiltration step can beemployed for larger fermentors (10 L to 40 L and larger). This step canbe performed utilizing Sartoriuos flat sheets with a pore size of 10K toa 5 fold concentration.

A capture step is performed with a Blue Sepharose 6 fast flow (GEhealthcare) equilibrated with 50 mM Tris-HCl/100 mM NaCl, pH 7. Thesupernatant was adjusted to 100 mM NaCl and passed through dead-endfilter (Whatman, Polycap TC) before loading to the column. The residencetime is maintained to 10 min with a 3 column volumes (CV) wash afterloading. The elution is performed in steps of 2 CV with 250 mM and 3 CVwith 1M NaCl. EPO elutes at the 1 M NaCl.

Macro-prep ceramic hydroxyapatite Type 140 μm (Bio-Rad) is used afterthe capture step. This column is equilibrated with 50 mM MOPS containing1M NaCl and 10 mM CaCl2 pH 7. 10 mM CaCl2 is added to the pooled EPOfrom the blue column before loading. The column wash is executed with 3CV of equilibration solution followed by 10 CV linear gradient from 0 to200 mM Na phosphate pH 7. EPO elutes between 60 mM and 100 mM Naphosphate.

Source 30S (GE Healthcare) can be used as an optional purification step.If this is the case, the pooled sample after hydroxyapatite is dialyzedagainst 50 mM NaAcetate pH 5 overnight at 4° C. and the column isequilibrated with the same buffer. A 10 CV linear gradient from 0 to 750mM NaCl is applied with EPO elutioning in between 350 to 500 mM NaCl.

Purified EPO Analysis

N-glycans were released from the purified EPO by treatment with PNGase F(Choi PNAS, Hamilton 2003 Science) and analyzed by SDS-PAGE according toLaemmli (Laemmli 1970) (FIG. 4).

The intactness and presence of aggregation of the purified EPO wasdetermined by size exclusion chromatography (SEC-HPLC) using a HitachiD7000 instrument with L7420 UV detector monitoring 280 nm, a WyattminiDAWN three-angle light scattering detector detecting at 690 nm, aWyatt Optilab rEx differential refractive index detector detecting at690 nm, a GE Healthcare Superdex 200 10/300 GL column (#17-5175-01), andWyatt ASTRA V 5.3.1.5 software (FIG. 4A-4D).

Ninety microliters of sample (>0.1 mg/ml) is placed in a sample vial,capped and then injected into the column. The HPLC gradient consists ofa 60 minute isocratic run at room temperature with a flow rate of 0.45ml/min using a buffer of 100 mM sodium phosphate (pH 6.8), 150 mM NaCl,and 0.05% sodium azide.

Molecular weights are calculated using a protein conjugate analysismodule of ASTRA software. The differential refractive index detector isset to the concentration detector. A dn/dc of 0.185 ml/g is used forprotein and 0.136 ml/g for PEG and glycans.

The purity of purified EPO was quantified by RP-HPLC using a HitachiD7000 HPLC instrument with L-7455 diode array detector and a Jupiter 5μ,C4 300 Å 150 mm×4.6 mm ID column (#00E-4167-E0, Phenomenex) (FIG. 4).Ninety microliters is injected and the sample is monitored at 280 nM andseparated according to the gradient below (column oven is pre-heated to80° C.) with the following buffers:

Buffer A: 0.1% trifluoroacetic acid (TFA) in HPLC-grade water spargedwith helium gas

Buffer B: 0.08% TFA in HPLC-grade acetonitrile sparged with helium gas

TABLE 2 Time Flow rate Buffer A Buffer B (min) (ml/min) (%) (%) 0 1 95 51 1 95 5 58 1 0 100 64.9 1 0 100 65 1 95 5 70 1 95 5

The quality of the hEPO protein was evaluated and post-translationalmodifications were assessed by peptide mapping (LC-MS) using an AdvionTriversa Nanomate, a Thermo Electron Finnigan LTQ mass spectrometer, aThermo Electron Finnigan Surveyor HPLC system, a Jupiter 4μ Peoteo 90Acolumn, 250×4.60 mm (#00G-4396-E0, Phenomenex), and a spin column with a10 kDa molecular weight cutoff (VS0101, Vivascience) (FIG. 7),

A 100 μl sample (>1 mg/ml) is placed in a 1.5 ml Eppendorf tube, 150 μl10M GuHCl and 2.5 μl 1 M Dithiothreitol (DTT) are added. The samplecontents are mixed and the sample is incubated for 1 hr at 37° C. Thesample is allowed to cool at room temperature and 10 μl 1M Iodoaceticacid (IAA) is added. Light is avoided by wrapping the sample tubes withaluminum foil and the samples are incubated at room temperature for 45min. The sample buffer is exchanged to 25 mM ammonium bicarbonate(NH₄HCO₃) pH7.8 (6 times for 10-fold dilution each time), and the finalvolume is reduced to ˜30 μl. 1 μg trypsin stock solution (constitutelyophilized trypsin in 50 mM acetic acid at 1 μg/μl and aliquot 20/tube,store at −20° C.) and acetonitrile to 5% (v/v) are added. The reactionsolution is placed at 37° C. and incubated overnight (˜16 hours).MALDI-TOF analysis is performed to ensure completion of the trypsindigest and the trypsin activity is inactivated. Formic acid is thenadded to a final concentration of 0.1% (v/v) to bring the pH down.Fifteen microliters of the digest is mixed with 15 μl HPLC buffer A(0.1% formic acid (FA) in HPLC-grade water) and loaded into a samplevial.

The HPLC setup is as follows:

1) Sample tray temperature: 4° C.;

2) Column oven temperature: 30° C.;

3) Partial loop injection;

4) UV detection: 215 nm and 280 nm;

5) HPLC gradient:

TABLE 3 Time Flow rate Buffer A Buffer B (min) (ml/min) (%) (%) 0 1 98 270 1 65 35 80 1 2 98 85 1 2 98 86 1 98 2 90 1 98 2The mass spectrometer setup is as follows:

1) Flow to LTQ ˜400n1/min through Advion Triversa Nanomate;

2) Capillary temperature: 115° C., capillary voltage: 5 v; tube lensvoltage: 77v

3) Scan set for neutral loss top 3 to MS4 for 90 min.

The chromatogram is plotted by looking at the base peak trace from themass detector. The peptides present in each peak are identified bysearching the hEPO sequence using Sequest software as well as manualinspection.

Example 5 Description of the PEG Molecules and Process Used forPEGylation

The following PEG molecules were used for PEGylation of hEPO:

40 kDa linear methoxy poly(ethylene glycol) proprionaldehyde from Dow(Cambridge, UK)

60 kDa linear methoxy poly(ethylene glycol) proprionaldehyde from Dow(Cambridge, UK)

30 kDa linear α-methyl-ω-(3-oxopropoxy), polyoxyethylene from NOFCorporation (Tokyo, Japan)

45 kDa branched2,3-Bis(methylpolyoxyethylene-oxy)-1-[(3-oxopropyl)polyoxyethylene-oxy]-propanefrom NOF corporation

The different activated PEGs (30 kDa, 40 kDa, or 60 kDa linear PEGs or45 kDa branched PEG) were added to the hEPO sample (conc. 1 mg/mL) in 50mM Sodium acetate buffer at pH 5.2 at a protein:PEG ratio of 1:10. Thereaction was carried out at room temperature under reducing conditionsby adding 10 mM sodium cyanoborohydride to the reaction mixture withovernight stirring. The reaction was stopped by adding 10 mM TRIS.Mono-PEGylated hEPO was purified using a SP sepharose Fast Flow columnand analyzed by SDS-PAGE (FIG. 5).

If further optimization is desired, the following parameters can beexamined:

1) Different pH ranges (pH 4.0, 4.5, 5.2 and 6.0)

2) Different molar ratio of hEPO:mPEG-aldehyde (1:5, 1:10, 1:20)

3) Room temperature versus 4° C.

4) Different types of PEGs

Example 6 Characterization of the PEGylated EPO Products

The four different PEGylated EPO products were analyzed by SEC HPLC(FIG. 6) as described in Example 4. For quantitation of N-linked glycanstructures (FIG. 7), N-linked glycans were released from the PEGylatedEPO by treatment with PNGase-F (Choi PNAS, Hamilton 2003 Science) andlabeled with 2-aminobenzidine (2-AB) using a commercial 2-AB labelingkit. HPLC was performed using a Prevail CHO ES, 5 micron bead,amino-bound silica column maintained at 30° C. The elution profile is asfollows (Solvent A: 100% acetonitrile, Solvent B: 50 mM ammonium formatepH 4.4):

TABLE 4 Flow rate Time/min (ml/min) % A % B 0 1 80 20 30 1 40 60 60 1 0100 65 1 0 100 70 1 80 20 80 1 80 20

FIG. 7 shows that approximately 70-74% of the PEGylated EPO products inthe tested samples are bisialylated (see the last peak in each panel).

Example 7 Formulation

A representative formulation of PEG-EPO may be 20 mM sodium phosphate,140 mM sodium chloride, 0.005% Polysorbate 80, pH 6.0, which is based ona similar marketed erythropoietin product. PEG-EPO can be formulated forinjection as a sterile, clear liquid, at multiple potencies, anddispensed in single dose glass vials ranging in concentration from25-500 μg/mL. As these concentrations are in the low range, there isconcern regarding protein adsorption to the vial, which could result inloss of dose. To minimize adsorption, surfactants are usually added toformulations (i.e., Polysorbate 20, Polysorbate 80) at limitedconcentrations. The need for surfactant will be based on materialcompatibility studies conducted during formulation development.

Other Formulations of Commercial EPOs

Commercial formulations of erythropoietin are known and my be adaptedfor use with the erythropoietins of the present invention. Some examplesof commercial EPO formulations are as follows:

ARANESP®: Polysorbate solution: Each 1 mL contains 0.05 mg polysorbate80, and is formulated at pH 6.2±0.2 with 2.12 mg sodium phosphatemonobasic monohydrate, 0.66 mg sodium phosphate dibasic anhydrous, and8.18 mg sodium chloride in water for injection, USP (to 1 mL).

Albumin solution: Each 1 mL contains 2.5 mg albumin (human), and isformulated at pH 6.0±0.3 with 2.23 mg sodium phosphate monobasicmonohydrate, 0.53 mg sodium phosphate dibasic anhydrous, and 8.18 mgsodium chloride in water for injection, USP (to 1 mL).

EPOGEN® is formulated as a sterile, colorless liquid in an isotonicsodium chloride/sodium citrate buffered solution or a sodiumchloride/sodium phosphate buffered solution for intravenous (IV) orsubcutaneous (SC) administration.

Single-dose, Preservative-free Vial: Each 1 mL of solution contains2000, 3000, 4000 or 10,000 Units of Epoetin alfa, 2.5 mg Albumin(Human), 5.8 mg sodium citrate, 5.8 mg sodium chloride, and 0.06 mgcitric acid in water for injection, USP (pH 6.9±0.3). This formulationcontains no preservative. Preserved vials contain 1% benzyl alcohol.

Example 8 In Vivo Analysis of PEG-EPO

In Vivo Studies in Mice:

Mouse efficacy study: The four different versions of PEG-EPO produced inP. pastoris strain YGLY3159 engineered to generate bi-antennaryterminally sialylated (>70%) human EPO were compared with commercialAranesp for their ability to increase hematocrit. C57B6 mice (age 7weeks at start of study, weight 18-20 g, 3 males/3 females per treatmentgroup) were obtained and acclimated for one week. Hematocrit values weredetermined before dosing to obtain a baseline for each animal. Animalswere segregated into six groups:

(1) vehicle (saline+100 μg/ml rHSA);

(2) ARANESP@2.5 μg/kg/dose (darbepoetin, albumin-free);

(3) PEG-EPO [40 kDa linear DOW]@ 2.5 μg/kg/dose;

(4) PEG-EPO [60 kDa linear DOW]@ 2.5 μg/kg/dose;

(5) PEG-EPO [30 kDa linear NOF]@ 2.5 μg/kg/dose;

(6) PEG-EPO [45 kDa branched NOF]@ 2.5 μg/kg/dose.

Animals were dosed by intraperitoneal injection twice weekly (Monday andThursday) for a total of five injections (dosing was stopped after 2.5weeks). The mice were bled weekly (Monday) and hematocrit values weredetermined. Mice injected with PEG-EPO conjugates of the presentinvention display an increase in hematocrit over commercial Aranesp. Thedata is presented in FIG. 8.

A second study was conducted in which the animals: C57 black mice (7weeks of age; ˜20 g; 3 males/3 females per group) were administered asingle weekly 2.5 μg/kg/dose of recombinant human epo. Injections wereon Thursdays and the animals were bled on Mondays during the course ofthe study. The study assessed the effect on hematocrit levels of onceweekly intraperitoneal administration of four PEG-EPO conjugates andAranesp (NESP). The data are presented in FIG. 13.

Immunogenicity Studies:

In order to address the immunogenic properties of PEG-EPO, rhesusmonkeys can be dosed subcutaneously twice/week for 2 weeks. AnELISA-based approach can specifically identify and measure antibodies torecombinant erythropoietin in rhesus and subsequently human blood.Following subcutaneous administration of PEG-EPO, serum samples can bemonitored over time for the generation of anti-erythropoietinantibodies. In addition, potential antibody responses can be correlatedwith pharmacokinetic and pharmacodynamic parameters that can bemonitored concurrently.

What is claimed is:
 1. A method for producing an erythropoietincomposition comprising the steps of: constructing a Pichia pastoris hostcell to attach pre-selected N-linked glycans to proteins, saidpre-selected N-linked glycans comprising greater than 25 mole percent ofan N-linked glycan having the structureN-acetylglucosamine₂Mannose₃N-acetylglucosamine₂Galactose₂N-acetylneuraminicacid₂ (GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂), and said host cell expressing asequence encoding erythropoietin operably linked to expression controlsequences recognizable by said host cell; culturing said transformedcell under conditions amenable to the expression of erythropoietin; andisolating said erythropoietin.
 2. The method of claim 1 wherein thePichia pastoris is ura5Δ::MET16 och1Δ::lacZ bmt2Δ::lacZ/KlMNN2-2,mnn4L1Δ::lacZ/MmSLC35A3 Δpno1Δmnn4Δ::lacZ met16Δ::lacZ,his1Δ::lacZ/ScGAL10/XB33/DmUGT, arg1Δ::HIS1/KD53/TC54,ADE1::lacZNA10/MmSLC35A3/FB8, PRO1::lacZ-URA5-lacZ/TrMDS1, AOX1:Shble/AOX1p/ScαMFpre-GFI800, TRP2::ARG1/MmCST/HsGNE/HsCSS/HsSPS/MmST6-33.3. The method of claim 1 wherein the Pichia pastoris is lacking theoch1, bmt2, mnn4L1, pno1, and mnn4 genes or gene functions and expressesMannosidase I, GlcNAc Transferase I, Mannosidase II, GlcNAc TransferaseII, UDP galactose transporter, UDP-galactose 4-epimerase, GalactosylTransferase, CMP-sialic acid transporter, UDP-GlcNAc2-epimerase/N-acetylmannosamine kinase, CMP-sialic acid synthase,N-acetylneuraminate-9-phosphate synthase, and Sialyl transferase.
 4. Themethod of claim 1, wherein greater than 50 mole percent of saidplurality of N-linked glycans consists essentially ofGlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.
 5. The method of claim 1, wherein greaterthan 75 mole percent of said plurality of N-linked glycans consistsessentially of GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂.
 6. The method of claim 1,wherein said GlcNAc₂Man₃GlcNAc₂Gal₂NANA₂ N-glycan is present at a levelfrom about 5 mole percent to about 80 mole percent more than the nextmost predominant N-linked glycan structure of said plurality of N-linkedglycans.
 7. The method of claim 1, wherein a polyethylene glycol moietyis linked to the N-terminal amino acid residue of at least 50% of theerythropoietin proteins, said link being an amine linkage.
 8. The methodof claim 7, wherein the polyethylene glycol moiety has a molecularweight of from about 20 kD to about 60 kD.
 9. The method of claim 7,wherein the polyethylene glycol moiety has a molecular weight of fromabout 30 kD to about 40 kD.
 10. The method of claim 7, wherein thepolyethylene glycol moiety is linear.